High affinity metal-oxide binding peptides with reversible binding

ABSTRACT

A dodecamer peptide, and its modified variant, having a repeating glycine-lysine sequence was created and found to bind with high affinity to oxide surfaces and certain activated polymeric surfaces. Reversible binding characteristics of the peptides were demonstrated. The peptides were integrated with proteins, cells and fusion proteins to provide attachment of the proteins, cells and fusion proteins to solid material structures. The peptides can be used to functionalize surfaces of components within mechanical, in mechanical, biomechanical, micro fluidic, electronic, bioelectronic, bio-optical, and biochemical devices. Experiments were carried out to assess functionalization and reusability of a suspended mass resonator&#39;s cantilever.

CROSS-REFERENCE TO RELATED U.S. APPLICATIONS

This application claims priority to U.S. Patent Application No.60/973,487 filed on Sep. 19, 2007, which is incorporated by reference.

GOVERNMENT FUNDING

The work described herein was conducted within a research programsupported in part with funding from the Army Research Office under grantnumber DAAD19-03-D-0004. The U.S. Government may have certain rights inthese inventions.

FIELD OF THE INVENTION

The inventive embodiments relate to engineered peptides which reversiblybind with oxide surfaces. The inventive embodiments further relate todevelopment of advanced compositions, materials and devices whichutilize engineered peptides bound to non-natural oxide surfaces.

BACKGROUND

The study of biomolecules for biotechnology applications has gainedwidespread interest in recent years and potentially offers utilizationof self-assembly and molecular recognition to develop and integrateadvanced biotechnological materials into the medical and high-technologyindustries. The ability of biomolecules to direct the growth andorganization of inorganic solids has been noticed in naturally-occurringbiomineralization systems. (E. Baeuerlein, Biomineralization: FromBiology to Biotechnology and Medical Application, Wiley-VCH, Weinheim,New York, 2000. S. Mann, Biomineralization: Principles and Concepts inBioinorganic Materials Chemistry, Oxford chemistry masters, 5, OxfordUniversity Press, Oxford, N.Y., 2001.) Natural biological systems haveevolved diverse structures, e.g., bones, teeth, mollusk shells andmagnetosomes, which exhibit greatly increased structural integritycompared to the organic scaffold from which they are formed. Naturalsystems can show exquisite control on the molecular scale, andbiomineralized materials can surpass their chemically synthesizedcounterparts in certain physical characteristics such as hardness,fracture resistance, and abrasion resistance. Certain advantages ofbiomineralizing systems can include spatial and temporal control overgrowth, remodeling mechanisms, and synthesis of mineral phases nototherwise possible at low temperature and pressure. (S. Weiner and L.Addadi, “Design strategies in ineralized biological materials,” J. ofMaterials Chem., Vol. 7 (1997) pp. 689-702.)

One step toward emulating natural biomineralizing systems is to developand identify certain biomolecules which can interact with, e.g., bindwith, reversibly bind with, non-natural inorganic materials. In acontrolled, laboratory environment, such biomolecules can be useful forthe development of advanced biotechnological materials and systems.

SUMMARY

Two peptides are created and disclosed which bind in a reversible mannerwith oxide surfaces. A first peptide, denoted K1 (SEQ ID NO: 1),comprises the sequence GKGKGKGKGKGK (SEQ ID NO: 1). A second peptide,denoted 2K1 (SEQ ID NO: 2), comprises the sequenceGKGKGKGKGKGKASGKGKGKGKGKGK (SEQ ID NO: 2). In various embodiments,either of the peptides can be bound to or attached to an oxide orplasma-activated surface. In various embodiments, a method for bindingeither inventive peptide to a surface comprises treating an oxide orplasma-activated surface with plural peptides wherein the peptides bindwith or attach to the oxide or plasma-activated surface. The step oftreating the oxide or plasma-activated surface can comprise exposing theoxide or plasma-activated surface to a solution containing aconcentration of plural peptides. In certain embodiments, peptides K1(SEQ ID NO: 1) or 2K1 (SEQ ID NO: 2) can be released from an oxide orplasma-activated surface. In various embodiments, a method for releasingbound peptides from a surface comprises exposing the bound peptides to asalt buffer so that the peptides release from the surface. The saltbuffer can have a selected level of ionic strength. In certainembodiments, peptides K1 (SEQ ID NO: 1) or 2K1 (SEQ ID NO: 2) can bereleased from a surface by subjecting the bound peptides to an electricfield or electrical bias. In various aspects, either inventive peptideK1 (SEQ ID NO: 1) or 2K1 (SEQ ID NO: 2) can be integrated withbiomolecules, antimicrobial peptides, proteins, fusion proteins,biomineralizing proteins, anti-analytes or cells.

Peptides K1 (SEQ ID NO: 1) and 2K1 (SEQ ID NO: 2) can further be used tofunctionalize oxide or plasma-activated surfaces, and in particularinorganic oxide surfaces. As an example, the peptides can be integratedwith proteins or cells and bound to oxide or plasma-activated surfaceson natural and non-natural materials. The bound constructs can beexposed to biomolecules which bind to the proteins or cells. In variousembodiments, the peptides mediate the binding of various biomolecules,e.g., antimicrobial peptides, proteins, fusion proteins, anti-analytemolecules etc., to oxide or plasma-activated surfaces. Thefunctionalized and/or bioactive surfaces can be incorporated in medical,electrical, magnetic, optical, biotechnology or high-technology devices.

The foregoing and other aspects, embodiments, and features of thepresent teachings can be more fully understood from the followingdescription in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

FIGS. 1A-1C depict biphasic binding behavior of the inventive peptides.The peptides 105 can be integrated with a host cell or protein 110.

FIG. 2A shows experimental results of yeast clones with integratedpeptide types of various residue grouping bound in PBS-BSAT to theA-(shaded) and R-(white) faces of sapphire. Each bar represents theaverage percent area coverage (P.A.C.) from two independent experimentsand includes error bars representing the standard deviation. Percentarea coverage (P.A.C.) is used as a measure of binding affinity.

FIG. 2B shows results for variant forms of the K1 (SEQ ID NO: 1) peptidebound in PBS-BSAT to the A-face of sapphire. Each data point representsthe average and standard deviation of binding from three independentexperiments.

FIGS. 3A-3B show experimental results for adhesion of MBP-K1 and MBP* tothe A-face of sapphire substrates performed in PBST buffer. ProteinMBP-K1 contains the K1 (SEQ ID NO: 1) peptide at the end of linker atthe c-terminus of maltose binding protein (MBP). Protein MBP* is thesame construct minus the twelve-amino acids of K1 (SEQ ID NO: 1).Adhesion is measured through a chromogenic reaction with horse radishperoxidase (HRP), which is conjugated to an anti-MBP antibody.Absorbance is measured using ultraviolet radiation at about 405 nm andsubtracted from absorbance measured for no-protein controls. (3A)Adhesion of MBP-K1 and MBP* to A-face sapphire at various dilutions ofthe incubation concentration. The arrow represents the peptideconcentrations used for the results reported in FIG. 3B. (3B) Adhesionof MBP-K1 and MBP* to the C-, A-, and R-faces of sapphire with anincubation concentration of about 10 μg/mL (0.23 μM). Each data pointrepresents the average and standard deviation of binding from at leasttwo independent experiments.

FIGS. 4A-4B show results from a modified ELISA in which peptides K1 (SEQID NO: 1) and 2K1 (SEQ ID NO: 2) were bound against sapphire andtissue-culture-treated polystyrene (TCT-ps). Peptides K1 (SEQ ID NO: 1)and 2K1 (SEQ ID NO: 2) were attached to the c-terminus of MBP. MBP-K1() and MBP-2K1 (▪) are tested against (4A) sapphire and (4B) TCT-ps.

FIGS. 5A-5B indicate dynamic binding characteristics of the inventivepeptide 2K1 (SEQ ID NO: 2). (5A) Association of MBP-2K1 to TCT-ps atthree separate concentrations (▪: about 22 nM; ▴: about 4.4 nM; : about0.9 nM) measured by enzymatic rates ([R]) of HRP conjugated to anti-MBPantibody. (5B) Dissociation of the same concentrations of MBP-2K1 fromTCT-ps after about 20 minutes association time. Solid lines representbest fits of the data to exponential rise and decay functions forassociation and dissociation data, respectively.

FIGS. 6A-6B demonstrate biphasic binding behavior of the inventivepeptide 2K1 (SEQ ID NO: 2) and its binding to a variety of oxidesurfaces. (6A) Demonstrates the effect of ionic strength on the bindingof MBP-2K1 to TCT-ps. (6B) Demonstrates binding of MBP-2K1 to variousoxide substrates. A: A-face sapphire; Z: Z-cut quartz (single-crystalSiO₂); T: thermally grown 100 nm-thick SiO₂ layer on Si wafer; S:standard microscope slide (borosilicate glass). Each data pointrepresents the average and standard deviation of binding from twoindependent substrates. For both experiments, absorbance at 405 nm wasmeasured after about a 10-minute incubation period in an ABTS reactionmix.

FIGS. 7A-7B depict methods for and results from surface micropatterningof affinity-tagged proteins. Two methods for patterning peptide-taggedproteins onto oxide surfaces are shown. (7A) For micro-plasma-initiatedpatterning (μPIP), a hydrophobic organic surface, such as polystyrene(PS), is masked with a PDMS mask in a desired pattern. The exposedsurface area of the PS is activated with a plasma. The mask is thenremoved and the affinity-tagged proteins preferentially bind to theactivated surface. Unbound proteins can be rinsed away. (7B) Formicrocontact printing (μCP), affinity-tagged proteins are first bound toan activated polydimethylsiloxane (PDMS) stamp that is topograpicallymicropatterned using soft-lithography techniques. The mask is thenplaced in intimate contact with a substrate. The protein can then bindto and be transferred to an oxide surface. (7A micrograph)Immuno-labeled MBP-2K1 is shown, imaged using a 40× objective lens,selectively bound to plasma-treated polystyrene (PT-PS), light area.Substantially no affinity-tagged proteins bind to the previously coveredarea, dark area. (7B micrograph) Immuno-labeled MBP-2K1 transferred totissue-culture-treated polystyrene using μCP. Affinity-tagged proteinsare transferred in the light area, where the PDMS was in intimatecontact with the substrate during the printing step. Substantially noproteins are transferred where the mask was not in contact with thesubstrate.

FIGS. 8A depicts electrochemical modulation of affinity-tagged proteinsbound to patterned indium tin oxide (ITO). Tagged proteins bound topatterned ITO electrodes (left) can be selectively desorbed byapplication of a positive electrical bias (right).

FIGS. 8B-8D are micrographs demonstrating electrochemical modulation ofaffinity-tagged proteins bound to patterned ITO. (8B) Immuno-stainedMBP-2K1 on patterned ITO electrodes and glass with a 5 minute incubationand no applied bias. (8C) Immuno-stained MBP-2K1 with a 5 minuteincubation under −0.3 V bias. (8D) Immuno-stained MBP-2K1 with a 30second incubation under +1.8 V bias. All microscope images were obtainedusing a 10× objective lens.

FIG. 9A is a pan-view illustration of a portion of a suspended massresonator (SMR) mass sensor. The SMR's cantilever 910 is shown whichcontains microfluidic channels. Affinity-tagged proteins 920 can beintroduced to and removed from the cantilever through the channels.

FIGS. 9B-9C show measured dose-response curves of (9B) MBP-2K1 and (9C)MBP* (MBP without the 2K1 (SEQ ID NO: 2) peptide) in 1× PBS binding tooxide surfaces in an SMR's cantilever. Protein in solution wasintroduced at time t=0. Flushing of the channels with high-salt bufferinitiated after about 6 minutes (arrows).

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings.

DETAILED DESCRIPTION

In overview, two engineered peptides were developed and identified tobind with oxide or plasma-activated surfaces. In certain embodiments,the inventive peptides serve as binding agents for proteins cells tooxide or plasma-activated surfaces which are non-native to certainbiological organisms and systems. Development of the peptides isdescribed briefly. Measurements of their binding affinity, and methodsfor reversible binding of the peptides are presented. Integration of thepeptides into advanced biotechnological systems is described.

I. Engineering of Peptides

An extensive study was carried out which led to the engineering of twopeptides which bind in a reversible manner to a variety of oxide andplasma-activated surfaces. Details of the study are reported in thedissertation by Eric M. Krauland entitled, “Towards Ration Design ofPeptides for Selective Interaction with Inorganic Materials,”Massachusetts Institute of Technology, Cambridge, Mass., September,2007, which is incorporated herein by reference. In brief, theinvestigations used a novel combination of yeast surface display (YSD),biopanning techniques, rational design methodology, and geneticengineering to arrive at two peptides having the desired properties of(1) binding to oxide or plasma-activated surfaces and (2) binding in areversible manner. In certain embodiments, using rational designprinciples in conjunction with the other techniques, peptides can beengineered that adhere cells or proteins to oxide surfaces at strengthsequivalent to those obtained for biopanning-enriched peptides whileexhibiting much lower compositional complexity than thebiopanning-enriched peptides.

One engineered peptide resulting from the study, denoted K1 (SEQ ID NO:1), is a twelve amino acid long peptide containing a repeatinglysine-glycine motif. Its sequence is represented as GKGKGKGKGKGK (SEQID NO: 1). The peptide carries a net charge of +6. This peptide canexhibit a binding affinity to a sapphire surface with a dissociationconstant of about 100 nanomolar (nM). The K1 (SEQ ID NO: 1) peptide canbe engineered from annealed oligonucleotides, e.g., G₆ peptide oligos,with BstXI compatible sticky ends ligated into BstXI sites of pBPZ.

In overview, the dodecamer peptide can be prepared by geneticallyengineering oligos into a yeast display vector. Briefly, complementaryoligos encoding the desired sequence and BstXI overhangs are annealedand then ligated into a BstXI digested pBPZ vector. Followingelectroporation into E. coli cells, Electromax DH10B (available fromInvitrogen, Carlsbad, Calif.), colonies are grown and DNA extracted forsequence confirmation. Correctly constructed vectors are thantransformed into EBY100 yeast using the Gietz quick transformationprotocol and maintained on glucose-based media (SD media).

A second peptide resulting from the study, denoted 2K1 (SEQ ID NO: 2),contains two K1 (SEQ ID NO: 1) peptides that are connected by analanine-serine linker sequence. Its sequence is represented asGKGKGKGKGKGKASGKGKGKGKGKGK (SEQ ID NO: 2). The peptide carries a netcharge of +12. This peptide can exhibit a binding affinity to a sapphiresurface with a dissociation constant of about 1 nM. The 2K1 (SEQ ID NO:2) peptide can be engineered in a manner similar to that used for the K1(SEQ ID NO: 1) peptide, although two K1 (SEQ ID NO: 1) sequences arelinked together with the alanine-serine spacer. In various embodiments,the amino acids used to link the K1 (SEQ ID NO: 1) peptides are selectedbecause the DNA that encodes for them includes an NheI restrictionenzyme site that can enable the original cloning strategy. Also incertain embodiments, the amino acids are selected because they encodefor one neutral amino acid (Ala) and one hydrophilic amino acid (Ser).

II. Binding of Peptides to Oxide Surfaces

In various embodiments, the structures of peptides K1 (SEQ ID NO: 1) and2K1 (SEQ ID NO: 2) facilitate their binding to oxide surfaces. Theinventors postulate that improved binding in certain peptides can arisefrom peptide geometries that allow maximal alignment of basic aminoacids towards a surface so that the charged groups within the peptidecan undergo local electrostatic interactions with the surface oxide. Invarious aspects, peptides K1 (SEQ ID NO: 1) and 2K1 (SEQ ID NO: 2) bindto a variety of oxide surfaces, e.g., sapphire, quartz, thermally grownoxide on silicon, amorphous borosilicate glass, and other oxides, athigh affinity and in a manner which can be selectively inhibited by highsalt conditions and/or exposure to an electric field. In certainembodiments, the inventive peptides also bind in a reversible mannerwith plasma-activated surfaces, e.g., various polymeric surfacessubjected to an oxygen plasma. In various embodiments, the peptide 2K1(SEQ ID NO: 2) and/or K1 (SEQ ID NO: 1) can be integrated with aselected protein, peptide, sequence or cell and function as a bindingagent to bind the selected protein, peptide, sequence or cell to atargeted substrate surface. As used herein, the term “integrated with”includes, without being limited to, fusion or binding of the peptidewith a protein, peptide, sequence or cell, as well as genetic expressionof the peptide within the protein, peptide, sequence or cell. It will beappreciated that targeting proteins to oxide, metal oxide, orplasma-activated surfaces with peptide tags such as 2K1 (SEQ ID NO: 2)or K1 (SEQ ID NO: 1) may provide a facile one-step alternative couplingchemistry for the formation of protein bioassays and biosensors.

A method for binding the peptides to an oxide or plasma-activatedsurface can comprise treating an oxide or plasma-activated surface withplural peptides, e.g., K1 (SEQ ID NO: 1) and/or 2K1 (SEQ ID NO: 2), orpeptide-tagged proteins or cells wherein the peptides bind or attach tothe surface. The step of treating the surface can comprise exposing thesurface to a solution containing the peptides or peptide-tagged proteinsor cells. The solution may be a saline solution having a selected levelof ionic strength. In certain embodiments, the ionic strength of thesolution can be between about 1 nanomolar (nM) and about 10 nM, betweenabout 10 nM and about 100 nM, between about 100 nM and about 1micromolar (μM), between about 1 μM and about 10 μM, between about 10 μMand about 100 μM, between about 100 μM and about 1 millimolar (mM), andyet in some embodiments, between about 1 mM and about 350 mM. The stepof treating the oxide or plasma-activated surface can further compriseproviding exposure of the surface to the solution for a selected periodof time, and under selected conditions, e.g., temperature and agitationof solution. The method for binding the peptides or peptide-taggedproteins or cells to a surface can further comprise removing the surfacefrom the solution containing the peptides after the selected period oftime. In certain embodiments, the duration of the selected period oftime and selected conditions can be chosen based upon a desired surfaceconcentration of peptides or peptide-tagged proteins or cells bound tothe surface.

In various embodiments, the peptides K1 (SEQ ID NO: 1) and/or 2K1 (SEQID NO: 2) can be integrated with a selected protein, peptide, sequence,or cell and function as a binding agent to bind the selected protein,peptide, sequence, or cell to a targeted substrate. In certain aspects,one or more peptide sequences K1 (SEQ ID NO: 1) and/or 2K1 (SEQ ID NO:2) can be transferred from yeast to a model protein. As an example, theK1 (SEQ ID NO: 1) or 2K1 (SEQ ID NO: 2) peptides can be integrated withthe maltose binding protein (MBP) by attachment to the end linker at theprotein's c-terminus. Further details of a MBP-K1 construct arepresented in Example 1. As another example, the K1 (SEQ ID NO: 1) or 2K1(SEQ ID NO: 2) peptides can be integrated with yeast cells bygenetically engineering oligos into a yeast vector. It will beappreciated that peptides which bind with high affinity to oxide orplasma-activated surfaces and can be integrated with a selected proteinor cell can serve as an affinity tag for facile protein, biomolecule, orcell immobilization. In certain embodiments, affinity-tagged proteins,biomolecules, peptides, or cells are utilized in modified enzyme-linkedimmunosorbant assay (ELISA) systems. In Example 2 below, a modifiedELISA is carried out with an affinity-tagged MBP to assess bindingaffinity of the tagged protein to certain oxide surfaces.

In certain embodiments, affinity-tagged proteins can serve as a basis tomake affinity-tagged fusion proteins. For example, the affinity-taggedprotein (MBP)-(2K1) can serve as a building block to create a variety ofaffinity-tagged fusion proteins. As an example, the affinity-taggedfusion protein (protein A)-(MBP)-(2K1) can be made from (MBP)-(2K1) andcan be used to immobilize certain antibodies to oxide surfaces. In suchan example, the protein A component can function as an anti-analyte andthe antibodies as analytes in a biochemical assay. In some embodiments,the affinity-tagged fusion proteins can be provided in an array, e.g.,patterned on an oxide substrate or disposed in multiple wells of amulti-well plate, useful for ELISA or immunoassays. As another example,the affinity-tagged fusion protein (bFGF)-(MBP)-(2K1) can be made from(MBP)-(2K1) and can be used in a bio sensor to monitor heparin contentin blood samples.

In some embodiments, affinity-tagged fusion proteins are created whichintegrate antimicrobial peptides (AmP's). As an example, anantimicrobial peptide can be linked to an affinity-tagged protein, e.g.,(AmP)-(MBP)-(2K1). In certain embodiments, the AmP is selected from thegroup consisting of: magainins, alamethicin, pexiganan, Template:MSI-78,Template:MSI-843, Template: MSI-594, Template:Polyphemusin, humanantimicrobial peptide, and Template:LL-37, defensins, and protegrins. Insome embodiments, other proteins, peptides, or sequences may besubstituted for the linking protein MBP. As an example, a peptide with asequence GGGGSGGGGSGGGGS (SEQ ID NO: 9) can be used in some embodimentsto link an inventive peptide K1 (SEQ ID NO: 1) or 2K1 (SEQ ID NO: 2) toan AmP. In certain embodiments, a linking peptide will be flexible andhave hydrophilic characteristics. The affinity-tagged antimicrobialfusion compositions can be attached to metal oxides or activatedpolymers and form antimicrobial peptide coatings. In variousembodiments, a metal oxide or activated polymer surface can befunctionalized with an affinity-tagged fusion composition and used toprevent bacterial adhesion and/or growth. In certain embodiments, theaffinity-tagged antimicrobial fusion compositions are adapted to provideantimicrobial coatings for medical devices.

In various embodiments, peptides K1 (SEQ ID NO: 1) and 2K1 (SEQ ID NO:2) can provide binding in a reversible manner to oxide surfaces. Incertain embodiments, the binding of the peptides to an oxide surface canexhibit biphasic behavior. As an example and referring to FIG. 1, apeptide 105 may be integrated with a host protein 110 wherein the hostprotein carries a net negative charge. When the peptide and host proteinare exposed to an oxide surface of a substrate 130 in a solution withlow ionic strength, charge repulsion between the host protein and oxidesurface can inhibit binding of the peptide to the surface. When thesolution has an intermediate ionic strength, long range charge screeningmay occur and permit binding of the peptide to the oxide surface. Athigh levels of ionic strength, short range charge screening can occurand inhibit binding of the peptide to the oxide surface. Biphasicbinding behavior can be advantageous in that is can allow for easyrefurbishing of substrates and sensors by incubation of bound peptidesin high-concentration salt buffers. Details and aspects of reversiblebinding by varying ionic strength are provided in Example 4.

Referring again to FIGS. 1A-1C in further detail, peptides 105 can beintegrated with a host protein or cell 110. In various embodiments, thepeptides 105 carry a net positive charge. In certain embodiments, thehost protein or cell 110 carries a net negative charge. In someembodiments, the host protein or cell 110 can carry a net positivecharge or be charge neutral. When ionic strength of a solution, withinwhich the peptide-tagged protein or cell is suspended, is low as shownin FIG. 1A, their can be insufficient free ions 120 to screen the hostnet charge from similar charge type on the surface of a substrate 130.In such a case, electrostatic repulsion can inhibit binding of thepeptide-tagged protein or cell to the substrate 130. When the ionicstrength of the solution is intermediate as depicted in FIG. 1B, freeions 120 can provide long-range charge screening and screen the netcharge of the host protein or cell and permit binding of the peptides105 to the substrate surface. At high ionic strengths, FIG. 1C,short-range charge screening can effectively screen all charges andinhibit peptide binding to the substrate surface.

A method for reversibly binding the peptides to an oxide surface cancomprise treating an oxide or plasma-activated surface with pluralpeptides, e.g., K1 (SEQ ID NO: 1) and/or 2K1 (SEQ ID NO: 2), orpeptide-tagged proteins or cells, wherein the peptides bind to the oxideor plasma-activated surface, and further subjecting the bound peptidesto a salt buffer having a selected ionic strength so that the peptidesrelease from the surface. The step of treating the surface can compriseexposing the surface to a solution containing the peptides orpeptide-tagged proteins or cells. The solution may be a saline solutionhaving a first level of ionic strength or salinity. The step of treatingthe surface can further comprise providing exposure of the surface tothe solution containing the peptides for a selected period of time, andunder selected conditions, e.g., temperature and agitation of solution.In various embodiments, the duration of the selected period of time andselected conditions can be chosen based upon a desired surfaceconcentration of peptides or peptide-tagged proteins or cells bound tothe surface. The step of subjecting the bound peptides or peptide-taggedproteins or cells to a salt buffer can comprise exposing the surface toa solution having a second level of ionic strength. The exposure of thebound peptides or peptide-tagged proteins or cells to the solutionhaving a second level of ionic strength can be carried out for aselected period of time, and under selected conditions, e.g.,temperature and agitation of solution. In various aspects, the secondlevel of ionic strength is greater than the first level of ionicstrength.

A first assessment of the binding affinity of the inventive peptides tooxide surfaces was carried out by the inventors using a yeast surfacedisplay (YSD) technique. In this study, peptides were expressed on thesurface of yeast cells, and adhesion to sapphire substrates wasmeasured. Additionally, the effect of charged residue placement alongpeptide sequences was evaluated. To this end, dodecamer peptides weredesigned and cloned onto the c-terminus of the Aga2 yeast displayconstruct. Each designer peptide, listed in Table 1, contained sixpositively charged amino acids but varied either in type of residue(lysine (K) or arginine (R)) or the grouping of these residues along thepeptide. Binding tests of the peptides against the A and R crystal facesof sapphire revealed two clear trends: First, lysine peptides boundbetter than identically constructed arginine peptides (e.g. K1>R1), andsecond, grouping charged residues decreased peptide binding (e.g.K1>K2>K3). These trends are evident from the measurements reported inFIG. 2A, which graphs percent area coverage (P.A.C.) of the bound yeastcells against the type of peptide. No clear preference for the A orR-face of sapphire was seen.

Two additional peptide variants of K1 (SEQ ID NO: 1) were designed andtested against the A-face of sapphire. The first, cK1 (SEQ ID NO: 6), isthe K1 (SEQ ID NO: 1) peptide flanked by cysteine residues that form adisulfide linkage in the oxidizing environment outside the cell andtherefore constrain the alternating lysine residues into a loop. Thispeptide showed binding comparable, if not slightly better, than theunconstrained K1 (SEQ ID NO: 1) peptide as seen in the graph of FIG. 2B.The second peptide, K1P (SEQ ID NO: 7), in which the second, fourth, andsixth glycine residues of K1 (SEQ ID NO: 1) were replaced with proline,showed a significant decrease in cell binding over K1 (SEQ ID NO: 1).

TABLE 1 Peptide sequence information. name sequence SEQ ID NO:net charge^(a) construct stop * n/a n/a Aga2, MBP X1 GXGXGXGXGXGX* 3 +6Aga2, MBP X2 GGXXGGXXGGXX* 4 +6 Aga2 X3 GGGXXXGGGXXX* 5 +6 Aga2 cK1CGKGKGKGKGKGKC* 6 +6 Aga2 K1P GKPKGKPKGKPK* 7 +6 Aga2 2K1 (GK)₆AS(GK)₆ 2+12 MBP ^(a)at neutral pH “*” denotes a stop codon “X” denotes eitherlysine (K) or arginine (R) Aga2: yeast surface display MBP: N-terminalmaltose binding protein

Although such cell-based experiments cannot resolve the detailedmolecular interaction, they can provide empirical evidence for theproposed interaction. From cell detachment assays, a clear trend isnoticed between the basicity of the peptide and the interactionstrength, which suggests the importance of multiple charge interactions.The importance of electrostatic interaction strength between peptide andsurface can be shown empirically by two experiments. First, an alteringof the ionic strength of a solution in which the peptides are suspended,which modulates electrostatic interactions by charge screening, canaffect peptide-mediated cell or protein binding. Second, an altering ofthe type of residue along the peptide can provide experimental resultssuggestive of the relevance of charge distribution along the peptide.For example, the difference between lysine and arginine binding indesigned peptides reported in FIG. 2A can result from differences in thelocalization of charge on the side-chain group. The positive charge inlysine is confined to a primary amine while in arginine it isdelocalized in the guanidinium group. This delocalization may weakensalt bridges with anions on the sapphire surface as compared to lysineand therefore result in a weaker binding interaction. The guanidiniumgroup can also become oxidized, which would eliminate the positivecharge, and disrupt electrostatic interaction with the surface. Inaddition, access of the positively charged groups to the surface may begreater in lysine than arginine due to a longer alkyl chain connectingthe peptide backbone to the charged moiety, as well as having a smallercharged moiety (amine versus guanidinium) in order to access surfacespecific grooves.

Without being bound by theory, the importance of side chain access tothe surface may be deduced empirically from binding experiments with thedesigned peptides. In a linear peptide chain, alternating amino acidside chain groups can orient in opposite directions. Therefore, inneighboring amino acids it is energetically unfavorable to align sidechains. Grouping of charged residues into neighboring amino acids, goingfrom sequence K1 (SEQ ID NO: 1) to K2 (SEQ ID NO: 4) to K3 (SEQ ID NO:5), would decrease the ability of peptides to align charged residues andmay result in the observed decrease in binding. As the basic amino acidsare grouped, it also leads to a decrease in charge coverage over thesurface. This decrease in surface coverage is not believed to be afactor because constrained peptide cK1 (SEQ ID NO: 6) bound to the oxidewith an affinity on the order of that found for K1 (SEQ ID NO: 1) whilebeing constrained to a smaller net surface area. (See FIG. 2B.)Additional evidence for the importance of charge alignment can be foundfrom the introduction of rigid kinks in the peptide backbone withproline residues, e.g., clone MP (SEQ ID NO: 7), and the resultingdecrease in peptide-mediated cell binding. From an engineeringperspective, the results of FIGS. 2A-2B are encouraging because theysuggests the ability to tune the affinity of a peptide by simplychanging the number of charged groups or the flexibility of thebackbone. In addition, the binding could be modulated by altering theionic strength of the buffer, an aspect explored in Example 4 below.

Although the inventive peptides show affinity for oxide surfaces, itwill be clear from the experiments and examples that the peptides canalso attach to activated surfaces. In certain embodiments, the peptidesbind with plasma-activated surfaces. In some embodiments,plasma-activated surfaces can be obtained by exposing a surface of amaterial to an oxygen plasma. In certain embodiments, polymeric surfacescan be activated by exposure to an oxygen plasma. As an example,surfaces of polymers such as polystyrene, polydimethylsiloxane,polyurethane, polycarbonate, and poly(methyl methacrylate) can beactivated in an oxygen plasma such that the inventive peptides attach tothese surfaces. In some embodiments, a polymeric surface coating may beapplied to a material, object or device, and the polymeric surfacecoating activated in an oxygen plasma so that the inventive peptidesattach to the surface coating. A polymeric surface coating may beestablished by methods of spin coating, dip coating, spray coating,vacuum depostion or similar coating techniques.

III. Integration of Peptides into Advanced Systems

The ability to coat surfaces with functional proteins, biomolecules, orcells can be useful for the construction and development of biosensors,bioassays and advanced biotechnological systems. Since oxide surfaces,such as SiO₂, or plasma-treated polymers, are common substrates inbiological applications, an oxide-binding peptide, such as 2K1 (SEQ IDNO: 2) or K1 (SEQ ID NO: 1), with an ability to adhere proteins withnanomolar affinity, may be a useful candidate as a versatile affinitytag. Methods of using the inventive peptides 2K1 (SEQ ID NO: 2) or K1(SEQ ID NO: 1) to create protein-functionalized oxide surfaces aredescribed. This section highlights several ways in which 2K1 (SEQ ID NO:2) can be used to immobilize proteins to oxide-surfaces. In particular,two methods of micro-scale patterning of 2K1-tagged MBP are described.The methods utilize soft-lithography techniques. Additionally, a methodto selectively inhibit tagged-protein from electronically-biasedsurface, e.g., indium tin oxide (ITO) electrodes, is described. Also,the utility of 2K1-tagged MBP in functionalizing a novel mass-basedbiosensor is demonstrated.

III-A. Micropatterning of Affinity-Tagged Proteins

The ability to functionalize surfaces on the micro-scale withbiologically active molecules has shown utility in biotechnical areasincluding stem cell biology (A. Khademhosseini, L. Ferreira, J.Blumling, J. Yeh, J. M. Karp, J. Fukuda, and R. Langer, “Co-culture ofhuman embryonic stem cells with murine embryonic fibroblasts onmicrowell-patterned substrates,” Biomaterials, Vol. 27, No. 36 (2006)pp. 5968-5977) and neurobiology (K. E. Schmalenberg and K. E. Uhrich,“Micropatterned polymer substrates control alignment of proliferatingschwann cells to direct neuronal regeneration,” Biomaterials, Vol. 26,No. 12 (2005) pp. 1423-1430) and immunology (J. Doh and D. J. Irvine,“Immunological synapse arrays: Patterned protein surfaces that modulateimmunological synapse structure formation in t cells,” Proceedings ofthe National Academy of Sciences of the United States of America, Vol.103, No. 15 (2006) pp. 5700-5705; H. Kim, R. E. Cohen, P. T. Hammond,and D. J. Irvine, “Live lymphocyte arrays for biosensing,” AdvancedFunctional Materials, Vol. 16, No. 10 (2006) pp. 1313-1323). Mostbiological microscale patterning techniques utilize a form of softlithography termed micro-contact printing (μCP). Initially developed forpatterning self-assembled monolayers by George Whitesides group (M.Mrksich and G. M. Whitesides, “Patterning self-assembled monolayersusing microcontact printing—a new technology for biosensors,” Trends inBiotechnology, Vol. 13, No. 6 (1995) pp. 228-235), it was first appliedto direct protein patterning in 1998 (A. Bernard, E. Delamarche, H.Schmid, B. Michel, H. R. Bosshard, and H. Biebuyck, “Printing patternsof proteins,” Langmuir, Vol. 14, No. 9 (1998) pp. 2225-2229; and C. D.James, R. C. Davis, L. Kam, H. G. Craighead, M. Isaacson, J. N. Turner,and W. Shain, “Patterned protein layers on solid substrates by thinstamp microcontact printing,” Langmuir, Vol. 14, No. 4 (1998) pp.741-744). In general, this approach involves (1) creating apolydimethylsiloxane (PDMS) elastomer “stamp” from aphotolithographically patterned silicon wafer, (2) adsorbing abiomolecule on the PDMS stamp, then (3) transferring the biolomoleculeto a desired substrate by contact printing. (R. S. Kane, S. Takayama, E.Ostuni, D. E. Ingber, and G. M. Whitesides, “Patterning proteins andcells using soft lithography,” Biomaterials, Vol. 20, No. 23-24 (1999)pp. 2363-2376) This general procedure was used to guide the developmenttwo schemes, depicted in FIGS. 7A-7B, for micropatterningaffinity-tagged proteins on oxide surfaces.

The first scheme, termed microscale plasma-initiated patterning (μPIP),involves first patterning the surface of a substrate using a mask and aplasma, and then incubating the substrate with a protein thatselectively attaches to the plasma-treated surface. (B. A. Langowski andK. E. Uhrich, “Microscale plasma-initiated patterning (mu pip),”Langmuir, Vol. 21, No. 23 (2005) pp. 10509-10514) This method, depictedin FIG. 7A, was used to bind MBP-2K1 to O₂-plasma-treated regions of apolystyrene substrate. Briefly, a PDMS mold 710 was used to maskselected regions of a polystyrene (PS) surface of a substrate 720. Next,the substrate was treated with an O₂-plasma, which oxygenates exposedregions into a highly electronegative hydrophilic surface. The PDMS moldwas then removed to expose both PS and plasma-treated polystyrene(PT-PS). Affinity-tagged protein MBP-2K1, which has nanomolar affinitytoward oxygenated PS, was then incubated with the substrates, e.g.,exposed in solution to the substrate. The substrate can then besubjected to a rinse or cleansing bath which removes unbound taggedproteins. A micrograph in FIG. 7A demonstrates adhesion ofimmuno-stained MBP-2K1 to a PT-PS region (light area) and substantiallyno adhesion of the tagged protein to an untreated PS region (dark area).Further details of μPIP with the inventive peptides are provided inExample 5.

In various embodiments, the μPIP method for micropatterningaffinity-tagged proteins on oxide surfaces comprises placing a PDMS moldor mask in contact with a surface of a substrate. In some embodiments,the mask may be a topographically-patterned mask or a stencil mask. TheμPIP method can further comprise exposing the substrate with contactedmask to a plasma, e.g., and oxygen plasma. The μPIP method can furthercomprise removing the PDMS mask, and incubating or exposing thesubstrate to a solution containing affinity-tagged proteins,biomolecules or cells. The substrate can be exposed to the solution fora selected period of time. The ionic strength of the solution can be aselected level. The μPIP method can further comprise rinsing thesubstrate with a cleansing rinse, so as to remove unboundaffinity-tagged proteins, biomolecules or cells from the substrate.

The μPIP method can benefit from certain optimizations. First, a PDMSmold or mask does not allow for facile patterning on the microscale. Ifa topographically-patterned mask is used, the short-lived plasma-inducedreactive species cannot penetrate at long ranges into microchannelsformed under the mask when placed in contact with the substrate.Although stencil masks can mitigate this problem, a need for supportstructures in stencil masks can limit available pattern shapes, e.g., anannulus shape can only be obtained by adding radial supports in astencil mask. Other lithographic techniques, which may aid patterning,have been described to patterned PT-PS features into PS. (J. B. Lhoest,E. Detrait, J. L. Dewez, P. V. deAguilar, and P. Bertrand, “A newplasma-based method to promote cell adhesion on micrometric tracks onpolystyrene substrates,” Journal of Biomaterials Science-PolymerEdition, Vol. 7, No. 12 (1996) pp. 1039-1054; S. A. Mitchell, M. R.Davidson, and R. H. Bradley, “Glow discharge modified tissue culturepolystyrene: role of surface chemistry in cellular attachment andproliferation,” Surface Engineering, Vol. 22, No. 5 (2006) pp. 337-344.)Additionally, the μPIP pre-patterning method is somewhat limited tomaterials that can be oxygenated or activated with plasmas or UV/ozonetreatment. Such materials comprise mainly carbonaceous polymers.However, given the inherent cell adhesion properties of plasma-treatedpolymers, specifically PT-PS over PS, e.g., as disclosed in (E. Detrait,J. B. Lhoest, B. Knoops, P. Bertrand, and P. V. D. de Aguilar,“Orientation of cell adhesion and growth on patterned heterogeneouspolystyrene surface,” Journal of Neuroscience Methods, Vol. 84, No. 1-2(1998) pp. 193-204; J. L. Dewez, J. B. Lhoest, E. Detrait, V. Berger, C.C. Dupont-Gillain, L. M. Vincent, Y. J. Schneider, P. Bertrand, and P.G. Rouxhet, “Adhesion of mammalian cells to polymer surfaces: fromphysical chemistry of surfaces to selective adhesion on definedpatterns,” Biomaterials, Vol. 19, No. 16 (1998) pp. 1441-1445; T. G. vanKooten, H. T. Spijker, and H. J. Busscher, “Plasma-treated polystyrenesurfaces: model surfaces for studying cell-biomaterial interactions,”Biomaterials, Vol. 25, No. 10 (2004) pp. 1735-1747), adding the abilityto selectively target proteins to the PT-PS with the inventive affinitytags should greatly increase the functionality of these systems.

The second patterning method shown in FIG. 7B is similar to traditionalmicrocontact printing (μCP) and has been used by the inventors topattern MBP-2K1 onto tissue-culture-treated polystyrene (TCT-PS).Briefly, a PDMS stamp is activated by exposure to 0₂-plasma andincubated with MBP-2K1. After incubation with protein and subsequentwashes, the stamp is pressed onto TCT-PS. At regions of contact betweenthe mask and substrate, the affinity-tagged protein can transfer fromthe mask to the substrate. A micrograph in FIG. 7B shows a TCT-PStreated surface immuno-stained for MBP. Two regions (light areas) ofprotein deposition are clearly observed flanking a protein deficient100-μm-wide channel (dark area). Variations of the procedure showed thatboth the peptide 2K1 (SEQ ID NO: 2) integrated with MBP and the PDMSactivation step are necessary for the efficient stamping of MBP-2K1 ontoTCT-PS. (Data not show for trials without 2K1 (SEQ ID NO: 2) and withoutPDMS activation.) Microcontact printing has been used to micropatternlaminin onto plasma-treated PMMA for the alignment and growth of Schwanncells. (K. E. Schmalenberg, H. M. Buettner, and K. E. Uhrich,“Microcontact printing of proteins on oxygen plasma-activatedpoly(methyl methacrylate),” Biomaterials, Vol. 25, No. 10 (2004) pp.1851-1857; and D. Y. Wang, Y. C. Huang, H. S. Chiang, A. M. Wo, and Y.Y. Huang, “Microcontact printing of laminin on oxygen plasma activatedsubstrates for the alignment and growth of schwann cells,” Journal ofBiomedical Materials Research Part B-Applied Biomaterials, Vol. 80B, No.2 (2007) 447-453.) Additional details of μCP with the inventive peptidesare provided in Example 6.

In various embodiments, a method for μCP patterning of oxide surfaceswith the inventive peptide-tagged proteins, biomolecules or cellscomprises providing a patterned PDMS stamp, and activating the surfaceof the stamp, e.g., by exposing it to plasma, so that the peptides bindwith the activated surface of the stamp. The method for μCP patterningcan further comprise incubating the PDMS stamp with affinity-taggedproteins, biomolecules or cells for a selected period of time, andsubjecting the PDMS stamp to a rinse which removed unbound proteins orcells. The method for μCP patterning can further comprise placing thePDMS stamp in contact with a substrate to be patterned, and leaving thestamp in contact with the substrate for a selected period of time. Themethod for μCP patterning can further comprise removing the PDMS stampfrom the substrate.

In general, printing methods involving plasma treatment can be easier toimplement, more cost effective, less labor intensive, and moreenvironmentally friendly than wet chemical methods. In addition, plasmaactivation of a PDMS stamp can decrease the protein equilibration timeover the more common hydrophobic PDMS surfaces. (A. Bernard, E.Delamarche, H. Schmid, B. Michel, H. R. Bosshard, and H. Biebuyck,“Printing patterns of proteins,” Langmuir, Vol. 14, No. 9 (1998) pp.2225-2229.) Hydrophobic adsorption to the stamps may also lead tounwanted denaturing of the protein of interest. (J. D. Andrade and V.Hlady, “Protein adsorption and materials biocompatibility—a tutorialreview and suggested hypotheses,” Advances in Polymer Science, Vol. 79(1986) pp. 1-63.) The additional functionality offered by the inventivepeptide affinity tags, such as 2K1 (SEQ ID NO: 2) or K1 (SEQ ID NO: 1),for selectively adhering to plasma-treated surfaces should greatlyexpand the repertoire of proteins suitable for μCP, including extremelysoluble proteins like MBP, without the requirement for long incubationtimes as required with hydrophobic surfaces.

III-B. Electronic Removal of Affinity-Tagged Proteins

Surfaces that respond to electrical stimulus can be useful for advancedsystems utilizing the inventive peptides, since they offer the potentialto electrically modulate peptide binding. Also they may enable facileintegration of advanced biotechnological systems which include theinventive peptides with well-established microelectronic devices. Incertain embodiments, microelectronic devices can provide superiortemporal resolution for the biotechnological systems. Recently, it hasbeen shown that poly-L-Lysine-grafted-polyethylene glycol (PLL-g-PEG)can be electrochemically desorbed from indium tin oxide (ITO) surfaces.(C. S. Tang, M. Dusseiller, S. Makohliso, M. Heuschkel, S. Sharma, B.Keller, and J. Voros, “Dynamic, electronically switchable surfaces formembrane protein microarrays,” Analytical Chemistry, Vol. 78, No. 3(2006) pp. 711-717.) ITO surfaces are potential candidates forbiosensors due to their low electrical resistivity and high opticaltransparency. (C. G. Granqvist and A. Hultaker, “Transparent andconducting ito films: new developments and applications,” Thin SolidFilms, Vol. 411, No. 1 (2002) pp. 1-5.) Although the mechanism ofprotein removal with applied positive bias is not currently understood,it has been hypothesized to be due to either, accumulated positivesurface charge repelling positively adsorbed molecules, or oxygengenerated from electrolysis that reacts with adsorbed molecules andchanges the physiochemical properties leading to adsorption. (C. S.Tang, et al.)

Given the oxide binding properties and presence of multiple lysineresidues in PLL and the affinity tag 2K1 (SEQ ID NO: 2), the inventorspostulated that a protein or cell tagged with the inventive peptides,e.g., 2K1-MBP, might be adapted for electrochemical desorption from ITO.FIGS. 8A-8D depict an experimental setup and results from 2K1-MBPdesorption experiments. Details of an experiment carried out with suchapparatus are presented in Example 7.

In various embodiments, ITO films on a glass substrate are etched topattern electrically isolated electrodes 810. Affinity-tagged proteins820 are then incubated with the ITO electrodes, while simultaneouslybiasing one or more electrodes. The bias can be applied for a selectedperiod of time and subsequently removed. The substrate can be extractedfrom the incubating solution and washed with a cleansing rinse whichremoves unbound affinity-tagged proteins, biomolecules or cells. Detailsand results from experiments carried out to demonstrate electrochemicalmodulation of surface attachment for the inventive peptides are providedin Example 7.

The ability to selectively adsorb affinity-tagged proteins ontoelectrode surfaces can be useful for the facile construction of proteinarrays. An advantage of using affinity-tagged proteins overPLL-g-PEG-biotin/Ni-NTA, is that fewer processing steps are involved todeposit proteins. For applications requiring large arrays or largenumbers of arrays, cutting the number of steps in one deposition cyclewould significantly decrease the overall processing time. Anotheradvantage is the possibility for lower applied potentials, due to thesmaller size of affinity tags like 2K1 (SEQ ID NO: 2) or K1 (SEQ IDNO: 1) versus PLL. Advantages of a PLL-g-PEG system over affinity-taggedproteins can be inherent anti-fouling properties of PEG, as well as thepotential to functionalize one PLL-g-PEG chain many times and thereforeincrease protein packing density. In some embodiments, a systemcomprising a combination of affinity-tagged proteins and PLL-g-PEG mayprovide faster and more versatile protein array construction.

III-C: Functionalization of Suspended Mass Resonators (SMR)

With advances in microfluidic and microfabrication techniques, there hasbeen a recent trend towards miniaturization of biosensors. (R. Raiteri,M. Grattarola, H. J. Butt, and P. Skladal, “Micromechanicalcantilever-based bio sensors,” Sensors and Actuators B-Chemical, Vol.79, No. 2-3 (2001) pp.115-126.) Advantages of these systems include highsensitivity, label-free sensing, small handling volumes, andparallelization. Recently, Burg et al. developed a microfabricated masssensor using a fluid-filled cantilever with reported mass sensitivitiesdown to approximately 300 attograms. (T. P. Burg, M. Godin, S. M.Knudsen, W. Shen, G. Carlson, J. S. Foster, K. Babcock, and S. R.Manalis, “Weighing of biomolecules, single cells and singlenanoparticles in fluid,” Nature, Vol. 446, No. 7139 (2007) pp.1066-1069.) A schematic of such a microfabricated sensing mechanism isdepicted in FIG. 9A. In various embodiments, the cantilever 910 of asuspended mass resonator (SMR) is driven to vibrate at its resonancefrequency and the vibrating cantilevered arm is monitored optically witha laser. As molecules are flowed into the cantilever, changes ineffective mass of the cantilever can be detected as a change in thearm's resonance frequency. An increase in mass due to accumulation ofadsorbed molecules on the walls, or the presence of large denseparticles, can alter the resonance frequency of the cantilever. Thealteration of resonance frequency can be detected by the opticalmonitoring system and provide a signal representative of a change inmass. In this manner, an SMR can measure adsorbed antibodies as well assingle gold and bacteria particles. (T. P. Burg, et al.)

Although a highly sensitive and versatile technique, a challenge forSMR, and other surface sensing techniques such as surface plasmonresonance (SPR) and quartz crystal microbalance (QCM), is the ability toperform facile and specific surface functionalization. Traditionaltechniques aimed at functionalization utilize covalent bonding methods,such as thiol linkages to gold or silanization of oxide surfaces.Although these techniques can form highly stable surfaces, these methodsare typically irreversible and therefore limit the versatility andlifetime of the device, especially for many microfabricated sensors,such as the SMR, in which the sensing surface is directly integratedinto the whole apparatus.

In one approach, PLL-g-PEG-biotin polymers were used to functionalize anSMR. The highly positively charged nature of PLL can bind to the nativesurface oxide of silicon in the cantilever of the SMR. This polymer hasbeen used to adhere biotinylated antibodies through a neutravidinintermediary layer. (T. P. Burg, et al.) Unfortunately, this processinvolves multiple steps that can be both time consuming and hard tostandardize.

Since the inventive peptides 2K1 (SEQ ID NO: 2) and K1 (SEQ ID NO: 1)can provide reversible binding with oxide or oxygen activated surfaces,the inventors postulated that they may be useful to biologicalfunctionalize an SMR and adapt it for reusable operation. Such an SMRcan be versatile in that is could be readily adapted to a variety ofmass sensing applications. In various embodiments, the functionalizationis stable over the course of an experiment, non-fouling, versatile inconferring specificity, and facile in preparation. In variousembodiments, functionalization with reusable operation is provided by aprotein, biomolecule or cell layer that contains a genetically encodedsurface-affinity peptide, wherein the protein, biomolecule or cell layeris reversibly bound to an oxide surface or activated surface within anSMR cantilever. An advantage to using the inventive peptides for SMR isthat they can provide non-covalent binding schemes to oxide surfaces inan SMR. Additionally, the functionalization process can be monitored andmodified in real time, e.g., modification can be achieved by changingionic strength of the solution and/or application of electricalpotentials. This can allow for faster optimization, better qualitycontrol, and more consistency between multiple functionalizations.Further, functionality can be added to the inventive peptide tagconstruct, e.g., protein fusion can be employed to add a specificreceptor or biomolecule for analyte sensing. As an example, protein Acan be added to the MBP-2K1 construct and reversibly bound to an SMR toprovide facile antibody capture and detection.

Experiments demonstrating functionalization of an SMR device werecarried out, and results are reported in Example 8.

IV. Applications

The inventive peptides can be used for a variety of biotechnologicalapplications. It will be appreciated that certain advantages of theinventive peptides suggest applications relating to material-specificbiomolecules. First, as described above, is an application as peptideaffinity tags for facile surface functionalization with proteins,biomolecules or cells. The inventive peptides can be integrated withproteins, biomolecules or cells and mediate binding to specificsurfaces. In some embodiments, the peptides can alleviate or eliminate aneed for prior chemical surface functionalization, and theirnon-covalent attachment characteristic can be useful for multi-purposesurfaces offering reusability. Such surfaces can include microfabricated sensors, such as the SMR, micropatterned surfaces, oxide orpolymeric surfaces, and bioseparation resins. In some embodiments, aninventive peptide can be integrated with or fused with amaterial-specific biomolecule, e.g., a biomineralizing protein. Thefused peptide-biomineralizing protein can be used to selectively directthe nucleation and growth of certain inorganic constructs onprepatterned or unpatterned substrates. Used in only two ways, asaffinity tags and nucleation tags, material-specific peptides have thepotential to impact fields as diverse as medicine, biotechnology, andelectronics.

Currently, only a few peptide affinity tags, such as the polyhistidinetag, directly recognize and bind with a solid support for proteinpurification systems. Identifying other tags that specifically recognizecommon materials such as silica and alumina can greatly increase theversatility of bioseparation systems while decreasing their cost. Incertain embodiments, the inventive peptides 2K1 (SEQ ID NO: 2) and/or K1(SEQ ID NO: 1) are used in separation and purification technologies. Asan example, the inventive peptides can be used in chromatography oraffinity-based purification systems. The inventive peptides can be usedas an alternative or supplement to hexa-histidine/nickel-basedseparation and purification systems.

Peptide-tagged proteins can be fused with other proteins, biomoleculesor cells for use in biochemical assays or biosensors. In variousembodiments, the fusion proteins, biomolecules or cells can be used toimmobilize other biomolecules, e.g., antibodies, analytes, antimicrobialpeptides, etc. The inventors have created two fusion proteins todemonstrate such applications. The fusion proteins created were (proteinA)-(MBP)-(2K1) and (bFGF)-(MBP)-(2K1). In certain embodiments, (proteinA)-(MBP)-(2K1) can be used to immobilize antibodies or analytes. Forexample, the affinity-tagged MBP of the fusion protein can bind to anoxide surface. The bound fusion protein can be exposed to a solutioncontaining a concentration of antibodies or analytes, e.g., hCG, whichcan bind with protein A. In certain embodiments, (bFGF)-(MBP)-(2K1) canbe used in a similar manner to monitor heparin content in blood samples.Both fusion proteins can be used in assay or biosensor devices, and thedevices may be reusable due to the reversible binding characteristics ofthe inventive peptides. For example, after an assay or biosensor hasbeen exposed to a target antibody, analyte, biomolecule, or cell, thebioactive oxide surfaces of the apparatus can be flushed with a highsalt solution to release bound peptides from oxide surfaces. In someembodiments, an electrical bias may be applied to the oxide surfaces toaid removal of bound peptides.

Another potential use of material-specific peptides is thesupramolecular organization of inorganic material on biologicalscaffolds. The ability to site-specifically place peptides at uniqueplaces on biological scaffolds can add versatility to supramolecularorganization. Aspects of such a technique have been demonstrated by theability to organize quantum dots or create nanowires on the sameorganism by utilizing different peptide attachment schemes. (S. W. Lee,C. Mao, C. E. Flynn, and A. M. Belcher, “Ordering of quantum dots usinggenetically engineered viruses,” Science, Vol. 296, No. 5569 (2002) pp.892-895; C. Mao, C. E. Flynn, A. Hayhurst, R. Sweeney, J. Qi, G.Georgiou, B. Iverson, and A. M. Belcher, “Viral assembly of orientedquantum dot nanowires,” Proc Natl Acad Sci USA, Vol. 100, No. 12 (2003)pp. 6946-6951.) Peptide-based nucleation on biological scaffolds mayalso prove useful in low energy, large-scale production of inorganicmaterials.

The inventive peptides can be used to attach biomolecules tobiologically directed architectures. Affinity-tagged fusion proteins canbe used, following the methods described above, to functionalize metaloxide surfaces of implantable medical devices. The inventive peptidescan be used, following the methods described above, to createantimicrobial surfaces by facilitating the attachment of one or moreantimicrobial peptides to an oxide surface. The inventive peptides canbe used to functionalize the surfaces of cell culture plates withbiomolecules. In some embodiments, the inventive peptides are used toattach biomolecules to certain components within electronic, mechanicalor chemical devices. For example, the peptides can be used to attachbiomolecules to solar cells, certain components of fuel cells, batteryelectrodes or charge storage media, transistors, magnetic or electronicmemory cells, or catalysts. In certain embodiments, the attachedbiomolecules and biologically directed architectures can improveoperability and/or durability of the device in which they areincorporated.

EXAMPLES Example 1 Peptide Mediated Adhesion of MBP

In order to demonstrate applicability of the inventive peptides asaffinity tags in protein bioassays, the inventors prepared two forms ofa maltose binding protein (MBP), one with and one without a c-terminusK1 (SEQ ID NO: 1) peptide, and investigated each form ###s binding tosapphire. Engineering and purification of MBP, a good model proteinbecause its stability in E. coli leads to large expression and highyield purifications, was accomplished using the pMAL expression kitavailable from New England Biolabs. Binding to sapphire was measured bythe activity of horse radish peroxidase (HRP) conjugated to anti-MBPantibody. The results of FIG. 3A show significantly improved binding(over several orders of magnitude) of the K1 (SEQ ID NO: 1) peptideintegrated with MBP (MBP-K1) over the naive MBP (MBP*) to the A-face ofsapphire. The binding of MBP-K1 was significantly more than backgroundbinding at about nanomolar concentrations and bound in similarquantities to MBP* but at about 500-1000 fold lower concentrations.Half-maximal binding for MBP-K1 occurred between about 10⁻⁷ and about10⁻⁶ M. Deriving a dissociation constant from this assay, however, isdifficult for a couple reasons. First, endpoint measurements ofabsorbance are prone to saturation non-linearities and therefore limitthe dynamic range of the signal. Second, assays were carried out inplastic wells which can exhibit intrinsic binding to the tested proteinsand thereby skew quantitative results. A modified ELISA can addressthese issues and results from modified ELISA experiments are presentedin Example 2.

The results of FIG. 3B show the selectivity of each sapphire crystalface for MBP-K1 over MBP* at an incubation concentration of about 10μg/mL (0.23 μM). All three faces demonstrate similar affinities for theprotein, again suggesting the lack of geometric specificity of thepeptides towards each surface. Interestingly, although the C-face bindsMBP* better than the other faces, it also shows an improvement inbinding the MBP-K1 protein. Such specificity could not be demonstratedwith yeast as endogenous yeast binding to the C-face masked anypotential peptide-specific binding.

For the experiments, the inventive peptides were cloned onto thec-terminus of MBP in the following manner. The pMAL-c2x vector(available from New England Biolabs, Beverly, Mass.), which encodes forthe cytoplasmic expression of MBP, was digested with EcoRI and HindIIIto allow for insertion of oligonucleotides on the c-terminus.Complementary oligos with EcoRI and HindIII compatible ends wereannealed and ligated into the digested pMAL-c2x vector. Ligationreactions were transformed into chemically competent TOP10 E. coli(available from Invitrogen, Carlsbad, Calif.) and cloning success wasverified through sequencing. Next, DNA from successful clones wastransformed into chemically competent TB1 E. coli for proteinexpression.

The procedures for protein expression and purification were taken fromthe pMAL Protein Fusion and Purification Kit (available from New EnglandBiolabs, Beverly, Mass.). Briefly, TB1 E. coli harboring the modifiedpMAL vectors were grown to mid-log phase in Glucose-Rich Media plusampicillin before induction with IPTG to a final concentration of about0.3 mM. After two hours of induction, cells were harvested bycentrifugation and frozen overnight at about −20 degrees. The cells werethen thawed in cold water and lysed by probe sonication. The crudeextract was separated from the insoluble cell mater by centrifugationand applied to an amylose resin column. The bound MBP constructs werethen eluted from the column with about 20 mM maltose in 1× column buffer(20 mM Tris HCl, 1 mM EDTA, 200 mM NaCl) and concentrated in 10000 MWCOCentricon Plus-20 centrifugal filtration devices (available fromMillipore, Billerica, Mass.). Purification steps were monitored bySDS-PAGE and the final concentration of protein was calculated byabsorbance at 280 nm and referenced with a known MBP standard from NewEngland Biolabs.

Purification of peptide 2K1 (SEQ ID NO: 2) was carried out as describedabove with noted exceptions. First, pelleted cells were resuspended andlysed in 1× column buffer supplemented with approximately 1M NaCl(CB-high salt). Branched, 2000 kDa polyethyleneimine (PEI) was thenadded to the crude extract at a final volume ratio of about 0.1%, inorder to elute and precipitate protein-bound nucleic acid. The crudeextract was then washed over an amylose column with CB-high salt beforeeluting as mentioned above.

Certain substrates for the binding experiments were provided as follows.Synthetic sapphire (α-Al₂O₃) windows were purchased from Crystal Systems(Salem, Mass.). The three orientations obtained were the C-plate (0 0 01), A-plate (1 1-2 0), and R-plate (1-1 0 2). The sapphire was producedby a heat exchanger method, cut with a tolerance of 2°, polished to an80/50 scratch/dig surface finish and a flatness of 10 waves per inch, asspecified by the manufacturer. The sapphire substrates were refurbishedfor multiple experiments by exposure to fresh piranha solution (3:1H₂SO₄: 30 wt % H₂O₂), followed by brief sonication in distilled water,and 70% (v/v) ethanol in water.

For this example, preparation and measurement of MBP bound to sapphiresubstrates was carried out as follows. Purified protein stocks wereserially diluted to the appropriate concentration in 1× PBS containing0.1% Tween20 (PBST). Approximately 250 μL, amounts of protein solutionwere added to clean sapphire substrates in 48-well plates and incubatedfor three hours under constant agitation on an orbital shaker.Substrates were washed twice, each time transferring to new wellscontaining about 400 μL, PBST and agitating for about 15 minutes.Substrates were then transferred to wells containing about a 2000-folddilution of stock HRP conjugated anti-MBP monoclonal antibody (availablefrom New England Biolabs, Beverly, Mass.) in PBS containing about 5mg/mL bovine serum albumin (PBS-BSA) and agitated for about 30 minutes.The substrates were washed two times as before with PBS-BSA thentransferred to a 96-well plate. Next, about 200 μL, of chromogensolution (0.5 mg/mL ABTS (2,2′-Azino-di-(3-ethylbenz-thiazoline sulfonicacid), 0.03% hydrogen peroxide in 0.1 M citrate buffer, pH 4.2) wasadded to the sapphire substrates under agitation. After about 15minutes, the substrates were removed and the absorbance of each well wasmeasured using ultraviolet radiation at about 405 nm wavelength (A405)on a 96-well UV/Vis plate reader (available from SpectraMAX 250,Molecular Devices, CA). Raw absorbance values were then subtracted froma background reading taken from substrates exposed to no MBP(approximately 0.2 arbitrary units (A.U.).

Example 2 Determination of Equilibrium Dissociation Constants

In order to obtain more quantitative binding affinity information, amodified ELISA was developed to assess the MBP binding to sapphire.First, sapphire substrates were incubated with tagged protein in 96-welluntreated polystyrene plates. Control experiments showed minimalbackground binding of proteins assayed to untreated polystyrene (unlikeplasma-treated polystyrene, which is discussed below). Second, theamount of anti-MBP-conjugated HRP adsorbed onto the surface was measuredby an enzymatic turnover rate rather than endpoint analysis, whichgreatly expanded the dynamic range of the assay. With these changes,modified MBP concentrations ([P]₀) were titrated over several orders ofmagnitude, and along with enzymatic rate ([R]), were used to findequilibrium dissociation constants (K_(D)) by fitting a simple twoparamter hyperbolic equation

$\begin{matrix}{\lbrack R\rbrack = \frac{{\lbrack R\rbrack_{\max}\lbrack P\rbrack}_{0}}{K_{D} + \lbrack P\rbrack_{0}}} & \left( {{EQ}.\mspace{14mu} 1} \right)\end{matrix}$

where [R]_(max) is the other free parameter representing the enzymaticrate at saturating levels of binding. EQ. 1 represents a Langmuir modelthat assumes first-order, reversible kinetics in which available proteinis not significantly depleted upon binding. This analysis was used todetermine the K_(D) of K1 (SEQ ID NO: 1) to sapphire and tissue culturetreated polystyrene (TCT-ps). TCT-ps, created by exposing polystyrene tooxygen plasma or UV/ozone. The plasma treatment can activate the surfaceand leave a net negatively charged, ionized surface. Plasma treatment isused as a surface treatment in tissue culture applications due to itsability to promote cell adhesion. In this study, plasma treatment wasused to provide a control amorphous surface for comparison with thestructured crystalline sapphire.

Equilibrium binding curves are shown in FIGS. 4A-4B and relevantparameters listed in Table 2. Enzymatic rates ([R]) are normalized tofree parameter ([R]_(max)) of EQ. 1 and best fits represented by thesolid lines. The equilibrium dissociation constant (K_(D)) was obtainedby fitting rate values to the two-parameter hyperbolic equation (EQ. 1)using the NLS fitting tool in OriginPro (available from OriginLab,Northhampton, Mass.). 95% confidence intervals are quoted for eachparameter based on the standard error times the critical value from thet-distribution at the given confidence value. The results show thatMBP-K1 attached to sapphire with a K_(D) of about 80 nM, which is infairly close agreement to values estimated from prior binding studiesreported in FIGS. 3A. MBP-K1 bound TCT-ps at K_(D) of about 360 nM, anapproximately five fold lower affinity than sappire.

The effect of increasing positively charged amino acids on bindingaffinity was tested by linking two K1 (SEQ ID NO: 1) sequences togetherusing an alanine-serine spacer. This peptide (2K1) was cloned onto thec-terminus of MBP and tested for binding to sapphire and TCT-ps (FIGS.4A-4B). Its binding affinity over a range of protein concentrations wascompared with that for MBP-K1. Doubling the length of K1 (SEQ ID NO: 1),increased the affinity towards sapphire 50 to100-fold, with an apparentK_(D) of about 1.3 nM. Similarly, binding to TCT-ps was dramaticallyincreased with apparent K_(D) of about 1.47 nM for MBP-2K1. Theseresults demonstrate that it can be possible to modulate the affinity ofthese peptides by altering the number of basic amino acids. Increasingthe multiplicity of peptides to increase affinity has been demonstratedby Tamerler et al., who report that tripling a selected gold-bindingpeptide into a 42 amino acid peptide increases equilibrium dissociationconstants to 10⁻⁷-10⁻⁶ M, with binding energies on the order ofself-assembled monolayers. (See C. Tamerler, E. E. Oren, M. Duman, E.Venkatasubramanian, and M. Sarikaya, “Adsorption kinetics of anengineered gold binding peptide by surface plasmon resonancespectroscopy and a quartz crystal microbalance,” Langmuir, Vol. 22, No.18 (2006) pp. 7712-7718.) The nanomolar affinities demonstrated by K1(SEQ ID NO: 1) and 2K1 (SEQ ID NO: 2) are surprisingly even greater thanthe 42 amino acid gold-binding peptide reported by Tamerler et al., andare on par with many antibodies.

TABLE 2 Best fit parameters to EQ. 1 from modified ELISA. peptide namesubstrate K_(D) (nM) [R]_(max) (A.U./min) K1 sapphire^(a) 80 ± 45 0.34 ±.05 TCT-ps^(b) 360 ± 4  1.14 ± .04 2K1 sapphire 1.3 ± 0.3 0.34 ± .01TCT-ps 1.47 ± 0.07 1.54 ± .03 R1 sapphire 460 ± 395 0.22 ± .05 TCT-ps130 ± 34  1.29 ± .06 K6 sapphire 320 ± 127 0.38 ± .05 TCT-ps 101 ± 23 1.23 ± .05 H6 Ni-NTA^(c) 30 ± 3  1.48 ± .06 ^(a)The A- and R-face ofsapphire combined. ^(b)Tissue-culture-treated polystyrene. ^(c)QiagenNi-NTA HisSorb plates.

By creating a His-tagged MBP clone, it was possible to compare theaffinity of the inventive designed sapphire binding peptides to acommonly used affinity tag for protein purification and immobilization,His-tag and nickle-NTA, under substantially similar assay conditions.Results are presented in Table 2. With a K_(D) of approximately 30 nM,the His-tag Ni-NTA interaction was between 2K1 (SEQ ID NO: 2) and K1(SEQ ID NO: 1). Concentrations of MBP-K1 at about 10⁻⁶ M that exhibitedclear binding to sapphire were similar to concentrations used forcapture of His-tagged proteins onto Ni-NTA covered plates. (D. Horakova,M. Rumlova, I. Pichova, and T. Ruml, “Luminometric method for screeningretroviral protease inhibitors,” Analytical Biochemistry, Vol. 345, No.1 (2005) pp. 96-101.) Therefore, it should be possible to use thesapphire affinity tags for similar protein immobilization applicationsas His-tags. An advantage of the sapphire tags over His-tags is the lackof surface chemistry necessary to modify substrates. Certain differencesbetween the two tags can be that the electrostatic mechanism for theinventive peptide binding may be less substrate specific and moresensitive to buffer ionic strength. Increasing the number of basic aminoacids to increase affinity may lead to a trade-off between affinity andspecificity, as a more basic peptide can have increased interaction withall electronegative surfaces.

In order to look more closely at the specificity of peptides towardssapphire, two more peptides were cloned onto the c-terminus of MBP: R1(SEQ ID NO: 3) and K6. The peptide K6 comprises the sequence GGGGGG *(SEQ ID NO: 8) and carries a net charge of +6. Expressed on yeast, R1(SEQ ID NO: 3) showed a significantly lower binding affinity towards Aand R-faces of sapphire (FIG. 2A), while K6 did not bind under similarassay conditions (data not shown). Consistently, each peptide expressedas MBP fusions also showed a reduced affinity (approximately 4-10 foldrelative to K1 (SEQ ID NO: 1)) to sapphire (Table 2). Interestingly,specificity to TCT-ps was reversed as both K6 and R1 (SEQ ID NO: 3)showed a greater affinity to TCT-ps (approximately 3-4 fold) than K1(SEQ ID NO: 1). These results suggest that lysine preferentiallyinteracts with the structurally organized sapphire surface anions (K1vs. R1) and that the ability to orient all basic groups in one directionis more important for crystalline surfaces than amorphous materials (K1vs. K6). However, further work is necessary to validate thesehypotheses.

Determination of dissociation constants with modified ELISA were carriedout in standard 96-well plates. Nickel-tri-nitroacetic acid binding wastested in Ni-NTA HisSorb plates (Qiagen). Tissue culture treatedpolystyrene binding was tested in BD-Falcon plates 35-3072. Sapphirebinding was tested in untreated polystyrene plates (Corning 3651) withresults averaged from both the A- and R-face. Protein stocks wereserially diluted in PBST, and about 200 μL amounts were added to thewells followed by about a 30 minute incubation. During all incubationsteps, plates were agitated at about 400 RPM on an orbital shaker. Wellswere rinsed three times with PBST and exposed to about 200 μL of 1:2000dilution of HRP conjugated anti-MBP monoclonal antibody as above.Following about a 30 minute incubation period with antibody, wells wereagain rinsed three times with PBST. In the case of sapphire binding,substrates were then transferred to new wells. Next, about 20 μL of 5mg/mL ABTS was combined with about 160 μL of 0.1 M citrate buffer, pH4.2, in each well. About 20 μL of 0.3% hydrogen peroxide was then addedand absorbance measurements were immediately taken every twelve secondsfor about four minutes to observe color production at 405 nm. The slopeof raw absorbance versus time was used to obtain rates of colorproduction (Abs_(405 nm)/min) and used as a metric for the amountanti-MBP antibody in the wells.

Example 3 Non-Equilibrium Association and Dissociation

Knowledge of the dynamics of peptide binding can provide valuableinformation for certain applications and further development of systemsutilizing the inventive peptides. Dynamics of peptide binding caninclude factors such as the time for the peptides to reach asteady-state binding equilibrium and the time for bound peptides to bereleased from a surface. Toward this objective, association anddissociation assays were performed with MBP-2K1 binding toTCT-polystyrene. Results from these assays are reported in FIGS. 5A-5B.Three concentrations of MBP-2K1 (about 22 nM, about 4.4 nM, and about0.9 nM) were chosen to bracket the previously calculated K_(D) of about1.5 nM, and then incubated with TCT-ps for various times up to abouttwenty minutes. FIG. 5A indicates that all three concentrations near asteady-state binding equilibrium after about twenty minute incubations.Approximate kinetic rate parameters k_(on) and k_(off) were determinedby fitting the data to a rising exponential using non-linear leastsquares regression. The fitting yields time constants for steady-stateassociation of about 0.0024 s⁻¹, about 0.0042 s⁻¹, and about 0.022 s⁻¹,for each respective higher concentration. Making the pseudo-first orderapproximation of non-depleting protein concentrations, the kinetic rateparameters k_(on) and k_(off) were estimated from the slope andintercept, respectively, from a linear fit of association time constantversus concentration. This resulted in a k_(on) of about 9.7×10 ⁵ M⁻¹s⁻¹and a k_(off) of about 0.0008 s⁻¹. From these values, it was possible tocalculate a non-equilibrium estimate of K_(D) of about 0.8 nM, which wasapproximately one-half of the measured equilibrium value of about 1.5nM.

FIG. 5B indicates dissociation dynamics of MBP-2K1 from TCT-ps for thesame three concentrations as used for the association study.Surprisingly, after 24 hours of washing, only about 20-30% of proteinwas removed from the surface. Decay time constants of about 1.8×10⁻⁶s⁻¹, about 2.2×10⁻⁶ s⁻¹, and about 4.6×10⁻⁶ s⁻¹ for each respectivehigher concentration, were determined by fitting the data and correspondto a decay half-life of approximately 60 hours. This data suggests thatpeptide 2K1 (SEQ ID NO: 2) is suitable for applications of proteinsurface labeling that can last up to several days. In some embodiments,the inventive peptides can provide surface labeling which persists forbetween about 1 hour and about 4 hours, between about 4 hours and about8 hours, between about 8 hours and about 16 hours, between about 16hours and about 30 hours, between about 30 hours and about 60 hours, andyet between about 60 hours and about 100 hours.

Interestingly, the decay constants from the dissociation experiment arealmost three orders of magnitude slower than the estimated k_(off) fromthe association experiment. This discrepancy might be explained by atwo-step binding event in which a loosely associated state transitionsto a more tightly bound state. Physically, the loosely bound state mightrepresent the protein domains interacting with the surface and the 2K1(SEQ ID NO: 2) peptide only partially bound, while the tightly boundstate could represent a rearrangement so that the protein domains becomeoriented away from the surface and the 2K1 (SEQ ID NO: 2) peptide morefully complexed with the surface. The dissociation assays might onlyprovide information about the tightly bound protein, as the looselyassociated state could either by removed or transitioned to tightlybound during the numerous rinses or 20 minute antibody incubation.Therefore, the results for the dissociation experiment FIG. 5B mightpertain primarily to the tightly bound state and yield a slow k_(off),while the association experiment can assess the flux between theunbound, loosely bound, and tightly bound proteins, and hence yield afaster estimated k_(off). Further experimentation with varyingincubation times followed by dissociation timecourses might verify thistwo-step hypothesis and parse out individual rate parameters.

The non-equilibrium association and dissociation experiments werecarried out using protein MBP-2K1 on tissue-culture-treated polystyrene(TCT-ps) 96-well plates (BD-Falcon plates 35-3072). For associationexperiments, protein stocks were diluted in PBST and about 150 μLamounts were added to the wells and incubated for a selected periods oftime (from about 20 minutes to about 15 seconds). During all incubationsteps, plates were agitated at about 400 RPM on an orbital shaker. Wellswere then rinsed two times with PBST and exposed to about 150 μL of1:2000 dilution of HRP conjugated anti-MBP monoclonal antibody asdescribed above. Following a approximately 20-minute incubation periodwith antibody, wells were again rinsed two times with PBST. The amountof HRP in each well was quantified as described in Example 2.Dissociation experiments were performed in a similar manner except thatabout a 20-minute protein incubation period was followed by washing thatvaried in time over a 24-hour period. Following a 20-minute incubationperiod with antibody, protein that remained bound was measured throughHRP enzymatic turnover. Approximate values for k_(off) were determinedby fitting to a decaying exponential function.

Example 4 Versatility of 2K1 (SEQ ID NO: 2) as a General ProteinAffinity Tag

Peptide 2K1 (SEQ ID NO: 2) was characterized further to assess itsutility as a general affinity tag. Results from these investigations arereported in FIGS. 6A-6B. FIG. 6A shows data representative of 2K1 (SEQID NO: 2) binding to TCT-ps substrates wherein the 2K1 (SEQ ID NO: 2) issuspended in various ionic strength solutions while exposed to thesubstrate. The protein was incubated at a final concentration of about0.1 μg/mL in 0.1× PBST supplemented with various concentrations of NaCl.The observed binding exhibits biphasic behavior. In this case, thedecrease in binding at low ionic strength can be due to repulsion of netnegative charge of the MBP protein (pI about 5 with −11 net charge atneutral pH) and the negatively ionized TCT-ps surface. Maximal bindingoccurs near an ionic strength of about 1× PBS (166 mM), which makes thetag suitable for bioassays performed at physiological conditions. Ationic strength greater than 350 mM, binding is almost eliminated and isbelieved to be due to charge screening between the peptides andsubstrate surface. A biphasic binding property can be advantageous inthat it should allow for facile refurbishing of substrates and sensorsby incubation with or exposure to high salt buffers.

In this study, synthetic sapphire was chosen as a model metal oxidesubstrate material. However, many applications can require differentoxide-based materials, so further experiments were carried out to assessbinding of the peptide to other oxide materials. Peptide 2K1 (SEQ ID NO:2) was tested for binding to a panel of oxide substrates, and resultsare shown in FIG. 6B. The inventive 2K1 (SEQ ID NO: 2) protein wasincubated at a final concentration of about 1 μg/mL in 1× PBST. Affinityof the peptide varied greatly on the various materials with a generaltrend of more structured substrates leading to more binding. Binding wassimilar between the A-face of sapphire and the C-face ofsingle-crystalline quartz (denoted with “Z” in the FIG. 6B), both highlyordered oxide surfaces. Exposure of the peptide to a less ordered oxide,thermally grown silicon dioxide (“T”) on a silicon wafer resulted inapproximately half the binding level observed for quartz. Binding toamorphous silica, in the form of a borosilicate microscope slide (“S”),was observed to be almost at background levels.

It is uncertain whether binding is directly related to atomic order, orwhether the atomic structure in these substrates just increased thelevel of surface anions (oxygen). The inventors propose the latterexplanation since amorphous TCT-ps does not exhibit long-range atomicorder but has been found to bind the peptides with high affinity. (See,for example, FIG. 4B.) In either case, the ability of 2K1 (SEQ ID NO: 2)to bind a variety of oxide materials increases its applicability as ageneral protein affinity tag.

Example 5 μPIP Patterning of MBP-2K1

Steps followed to micropattern a substrate by the method of μPIP aredepicted in FIG. 7A. A PDMS mask was cleaned by sonication in 70% (v/v)ethanol in water for about 20 minutes and then allowed to dry in air. Apolystyrene substrate was cleaned with three rinses of warm soapy waterfollowed by three rinses with ethanol and ultra-pure water before dryingin air. The PDMS mask was placed on polystyrene and the pair exposed tooxygen plasma for about one minute. The PDMS mask was then removed fromthe polystyrene substrate, and a solution of about 10 μg/mL MBP-2K1 inPBST was applied to the polystyrene surfaces for about 20 minutes. Thepolystyrene substrate was washed three times with about 1 mL PBST. Next,about 1 mL of anti-MBP mouse IgG, diluted 1:2000 from a 1 mg/mL stock(New England Biolabs) in PBS-BSA was incubated for about 30 minutesbefore washing three more times with about 1 mL PBST. A second 30 minuteantibody labeling step was performed with about 1 mL of 10 μg/mLgoat-anti-mouse IgG-AlexaFluor488 in PBS-BSA, followed by an additionalthree washes. Substrates were then imaged using fluoresceinisothiocyanate (FITC) filter sets and standard fluorescent microscopytechniques.

A micrograph of a region of a substrate patterned with theaffinity-tagged proteins by the method of μPIP is shown in FIG. 7A. Thelight-shaded areas are regions where the tagged proteins are attached tothe substrate. The dark-shaded area is a region where substantially notagged proteins are bound to the substrate.

Example 6 μCP Patterning of MBP-2K1

Steps followed to micropattern a substrate by the method of μCP aredepicted in FIG. 7B. A topographically-patterned PDMS stamp was cleanedby sonication in 70% (v/v) ethanol in water for about 20 minutes andthen allowed to dry in air. The PDMS stamp was then exposed to oxygenplasma for about one minute followed by about one minute exposure toambient air. A 10 μg/mL MBP-2K1 solution in PBST was exposed to thestamp for about 15 minutes. Excess and unbound protein was washed offthe stamp by submersing PDMS in ultra-pure water 3 times followed bydrying under a nitrogen air stream. The stamp was then pressed ontotissue-culture-treated polystyrene substrate (TCT-ps; BD-Falcon 6-wellplates, 35-3046) and left in contact for about 20 minutes. The stamp wasthen removed, and the substrate stained with primary and secondaryantibodies as described in Example 5.

A micrograph of a region of a substrate patterned with theaffinity-tagged proteins by the method of μCP is shown in FIG. 7B. Thelight-shaded areas are regions where the tagged proteins are attached tothe substrate. The dark-shaded band is a region approximately 100microns wide where substantially no tagged proteins are bound to thesubstrate. The fidelity of the line edges suggest that the patterningresolution is better than about 10 microns.

Example 7 Electronic Removal of Affinity-Tagged Proteins

An experiment was carried out to investigate electrochemical desorptionof affinity-tagged proteins (MBP-2K1) from indium tin oxide (ITO)substrates. Glass substrates with a thin film of ITO were obtained fromDelta Technology (resistivity about 13-30 Ω-cm). To create patterned ITOelectrodes on the substrates, silicon tape was used as a patterningmask. Unmasked ITO was etched away to expose the underlying glasssubstrate by exposure to agua regia (100 mL H₂O, 80 mL hydrochloricacid, 35 mL nitric acid) for about 15 minutes followed by several rinsesin ultra-pure water. After removal of the silicon tape, the substrateand patterned ITO electrodes were cleaned in piranha solution (3:1H₂SO₄: 30 wt % H₂O₂) for about 10 minutes, followed by brief sonicationin ultra-pure water, and in 70% (v/v) ethanol in water. Clean ITOsurfaces were connected to a voltage source with an aluminum adhesiveelectrode and wells were created on the clean ITO/glass substrates with1-cm-diameter silicone gaskets (holding volume about 0.2 mL). About 180μL of PBST was added to the well and Pt and Ag counter and referenceelectrodes, respectively, were lowered into the well approximately 1 mmbelow the top of the meniscus. Next, about 20 μL, of 10 μg/mL MBP-2K1was mixed into the well by pipette, followed by application of anelectrical bias for a selected period of time. After incubation underelectrical bias, the well was washed three times with PBST followed byantibody staining as described in Example 5, with all volumes adjustedto about 200 μL. Aspects of the experiment are depicted in FIG. 8A.

The substrate surfaces were imaged using a microscope to determine thepresence of MBP-2K1. As shown in FIGS. 8B-8C, applying a 0 V and about a−0.3 V bias for about 5 minutes to the ITO during incubation did notsubstantially alter the binding of MBP-2K1 versus the unbiased ITOelectrode. Conversely, applying about +1.8 V bias to one electrode foronly about 30 seconds, FIG. 8D, significantly inhibited binding ascompared to the unbiased and −0.3V biased cases. Similar conditions wereused by Tang et al. to selectively remove PLL-g-PEG from ITO electrodes,although they reported significant electrolysis and degradation of theelectrode at a bias of +1.8 V, which was not observed in theseexperiments. The discrepancy might be attributed to certain differencesin electrochemical cells used. Conversely, electrolysis and destructionof the ITO electrode was seen at similar negative potentials andtherefore large negative biases could not be tested.

Example 8 Functionalization of an SMR with Peptide 2K1 (SEQ ID NO: 2)

In view of results showing protein binding to thermally grown SiO₂ onsilicon, FIG. 6B, peptide 2K1 (SEQ ID NO: 2) was selected to investigatereversible protein functionalization of a suspended mass resonator (SMR)mass sensor. An SMR was fabricated and operated as described in T. P.Burg, A. R. Mirza, N. Milovic, C. H. Tsau, G. A. Popescu, J. S. Foster,and S. R. Manalis, “Vacuum-packaged suspended microchannel resonant masssensor for biomolecular detection,” Journal of MicroelectromechanicalSystems, Vol. 15, No. 6 (2006) pp. 1466-1476, and T. P. Burg, M. Godin,S. M. Knudsen, W. Shen, G. Carlson, J. S. Foster, K. Babcock, and S. R.Manalis, “Weighing of biomolecules, single cells and singlenanoparticles in fluid,” Nature, Vol. 446, No. 7139 (2007) pp.1066-1069. The cantilever portion of the SMR is depicted in FIG. 9A.Before application of protein to the SMR, stock solutions of MBP-2K1 andMBP* were buffer exchanged into 1× PBS with Zeba Desalt Spin columns(available from Pierce), followed by filtration through Ultra Free-MC0.22 μm filters (available from Millipore). After cantilever surfacecleaning with piranha solution, an experimental run consisted of thefollowing steps: (1) equilibration of the sensor in 1× PBS runningbuffer, (2) injection of the protein using a bypass channel in the SMR,(3) measurement of cantilever resonance frequency, (4) washout ofunbound protein with buffer, and (5) release of protein from oxidesurfaces within the fluidic channels with a flushing rinse of high-saltbuffer (0.1 M phosphate buffer, 1 M NaCl). Raw data was collected asdescribed in Burg et al., processed, and reported. Briefly, theprocessing comprised adjusting the raw data by subtracting out spikes invibrational frequency due to variations in solution density betweenrunning buffer and protein solutions, and then normalizing the data tothe pre-protein resonance frequency.

The results of FIGS. 9B-9C show specific adhesion of peptideaffinity-tagged protein MBP-2K1 to the SMR cantilever. The cantileverwas saturated with MBP-2K1 at an incubation concentration of about 100μg/mL, and the bound protein resulted in about a 6 Hz frequency shift ofthe cantilever's resonance frequency. Conversely, MBP without 2K1 (MBP*)caused less than about a 1 Hz shift at a ten-fold higher concentrationof about 1 mg/mL (FIG. 9C). This experiment and results demonstrate thepotential of 2K1 (SEQ ID NO: 2) as an affinity tag for reversiblefunctionalization in an SMR. Further experiments could optimize bufferconditions, concentrations, and incubation times to ensure consistentdeposition of functionalized layers within the device.

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings have been described in conjunctionwith various embodiments and examples, it is not intended that thepresent teachings be limited to such embodiments or examples. On thecontrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made by one of ordinary skillin the art without departing from the spirit and scope of the appendedclaims. All embodiments that come within the spirit and scope of thefollowing claims and equivalents thereto are claimed.

1. A peptide having the sequence GKGKGKGKGKGK (SEQ ID NO: 1) or GKGKGKGKGKGKASGKGKGKGKGKGK (SEQ ID NO: 2).
 2. A method comprising treating an oxide or plasma-activated surface with plural peptides of claim 1 wherein the peptides bind with the oxide or plasma-activated surface.
 3. The method of claim 2 further comprising exposing the bound peptides to a salt buffer so that the peptides release from the oxide or plasma-activated surface.
 4. The method of claim 2 wherein the peptides are integrated with biomolecules.
 5. The method of claim 2 wherein the peptides are integrated with antimicrobial peptides.
 6. The method of claim 2 wherein the peptides are integrated with proteins.
 7. The method of claim 2 wherein the peptides are integrated with fusion proteins.
 8. The method of claim 2 wherein the peptides are integrated with cells.
 9. The method of claim 2 wherein the peptides are integrated with biomineralizing proteins.
 10. The method of claim 2 wherein the oxide or plasma-activated surface comprises a surface of an implantable medical device.
 11. The method of claim 2 wherein the oxide surface comprises a metal oxide surface of an implantable medical device.
 12. (canceled)
 13. The method of claim 2 wherein the oxide or plasma-activated surface comprises a surface of a cantilever of a suspended mass resonator or a surface within a chromatography or affinity-based purification apparatus.
 14. (canceled)
 15. The method of claim 2 wherein the oxide or plasma-activated surface comprises a surface of a component selected from a group consisting of a solar energy device, a fuel cell, a battery, a transistor, a memory component, a surface of a catalyst, a surface of a biosensor, and an electrically conductive surface. 16.-21. (canceled)
 22. The method of claim 2 wherein the oxide surface is indium tin oxide.
 23. The method of claim 2 wherein the plasma-activated surface comprises polystyrene, polydimethylsiloxane, polyurethane, polycarbonate, or poly(methyl methacrylate) subjected to an oxygen plasma.
 24. A fusion protein comprising the peptide as claimed in claim 1 and maltose binding protein.
 25. The fusion protein of claim 24 wherein the peptide is attached to an oxide or plasma-activated surface.
 26. The fusion protein of claim 24 additionally integrating an antimicrobial peptide.
 27. The fusion protein of claim 24 further comprising an anti-analyte bound to the maltose binding protein.
 28. The fusion protein of claim 27 wherein the peptide is attached to an oxide or plasma-activated surface.
 29. The fusion protein of claim 27 wherein the anti-analyte is protein A or bFGF. 30.-60. (canceled) 